Process for the production of glucose isomerase

ABSTRACT

An improved process for the production of glucose isomerase involves the addition of a specific amount of glycine (aminoacetic acid) to the fermentation medium. This increases the amount of the enzyme, glucose isomerase, that is produced by the microorganism, which preferably is a strain of Streptomyces, most preferably, S. olivochromogenes ATCC 21,114. Even further improvement is obtained by also adding a small amount of ammonium nitrate to the culture medium.

United States Patent [191 Heady et al.

[ 1 Nov. 6, 1973 PROCESS FOR THE PRODUCTION OF GLUCOSE ISOMERASE [75]Inventors: Robert E. Heady, Park Forest;

William A. Jacaway, Jr., Downers Grove, both of III.

[73] Assignee: CPC International, Inc., Englewood Cliffs, NJ.

[22] Filed: May 20, 1971 [21] Appl. No.: 145,351

[52] US. Cl. 195/66 R, 195/31 F, 195/114 [51] Int. Cl ClZd 13/10 [58]Field of Search l95/31 F, 61, 62,

[56] References Cited UNITED STATES PATENTS ll/1971 Iizuka et al. 195/31R OTHER PUBLICATIONS Danno, G., Agr. Biol. Chem. Vol. 34, p.1,658-l,667,

Yoshimura et al., Agr. Biol. Chem. Vol. 30, p. LOIS-1,023, 1966.

Primary Examiner-Alvin E. Tanenholtz Attorney-Frank E. Robbins, John B.Goodman, Janet E. Price, Joseph Shekleton, Dietmar H. Olesch and DorothyR. Thumler [57] ABSTRACT 5 Claims, 3 Drawing Figures PROCESS FOR THEPRODUCTION OF GLUCOSE ISOMERASE FIELD OF THE INVENTION This inventionrelates to an improved process and culture medium for the production ofan enzyme preparation containing glucose isomerase.

DESCRIPTION OF THE PRIOR ART Since Marshall and Kooi published theinformation about the first technically feasible dextrose-levuloseenzymatic isomerization process in Science, Apr. 5, 1957, Vol. 125, No.3249, pp 648-649, many attempts have been made to develop commerciallyfeasible enzymatic isomerization processes.

OBJECTS OF THE INVENTION One object of the present invention is toprovide an improved process for the production of a glucose isomeraseenzyme preparations.

A more specific object of the invention is to provide a practicalprocess for the production of a glucose isomerase enzyme preparation byimproving the composition of the culture medium.

A related object of the invention is to provide improved culture mediafor the growth of microorganisms that produce glucose isomerase.

Other objects of the invention will appear here and after from thespecification and from the recitals of the attended claims.

SUMMARY OF THE INVENTION We have found an improved method for theculture of the cells of microorganisms that produce glucose isomeraseenzyme preparations. This method comprises culturing the cells in amedium comprising glycine, in an amount that enhances the production ofthe enzyme. While this amount may be up to 0.5 g. of glycine per 100 ml.of the culture medium, in practice there is little real advantage toadding an amount of glycine in excess of 0.3 g. per 100 ml. of theculture medium. For the sake of economy,.it is preferred that the amountof glycine added to the culture medium be in the range from 0.1 g. to0.25 g. of glycine per 100 ml. of the culture medium.

In addition, we have discovered that the presence of a small amount ofammonium nitrate in the culture medium produces a synergistic effectwith the glycine in enhancing the production of the enzyme. Generally,the amount of ammonium nitrate need not exceed 0.5 g. per 100 ml.,although more than this may be used in combination with the glycine,without any material ill effect. However, no real advantage is obtainedwhen the amount of ammonium nitrate present exceeds 0.5 g. per 100 ml.,and generally, it is preferred that the amount of ammonium nitrate fallin the range from 0.1 g. to 0.3 g. per 100 ml. of the culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings:

FIG. 1 is a graphical representation showing the effects of additions ofglycine by itself, of ammonium nitrate by itself, and of combinations ofglycine and ammonium nitrate at different rates of addition, to aparticular culture medium, in terms of enzyme production in units perml. of culture medium, in accordance with one embodiment of the presentinvention;

FIG. 2 is a graphical representation of the effects of additions ofglycine,'of ammonium nitrate, and of combinations of glycine andammonium nitrate, to the culture medium, on cell production in terms ofcell weights in grams per liter, dry basis, and

FIG. 3 is a graphical representation of the effects of additions ofglycine by itself, of ammonium nitrate by itself, and of combinations ofglycine and of ammonium nitrate together, to the culture medium, interms of the specific activity as measured in units of isomerase pergram of cells, dry basis.

GENERAL DESCRIPTION OF THE INVENTION The process of the presentinvention can be employed, so far as is known, for the production of alltypes of glucose isomerase enzyme preparations, including those wherethe enzyme preparation has a dominant or more rapid catalytic action onan isomerization other than that of glucose to levulose. These enzymepreparations can be derived from a large number 7 of different microbialsources.

Each enzyme preparation seems to have its own particularcharacteristics, such as, for example, optimum pH, optimum temperature,the required metal ions, Michaelis constant, and the mechanism oflevulose formation, all of which seem to be somewhat different from oneenzyme preparation to another. However, the process of the presentinvention seems to be applicable to the production of glucose isomeraseenzyme preparations from all known microbial sources, and morespecifically, from all Streptomyces species and strains and all Bacillusspecies and strains that produce glucose ganisms have been deposited inthe American Type Culture Collection, Washington, D.C., and added to itspermanent collection of microorganisms. They have been assigned thefollowing identification: S. venezuelac ATCC 2l,1 l3 and S.olivochromogenes ATCC 21,114.

The enzyme preparation is produced in the usual manner. An inoculumprepared, for example, on an agar slant, is used to inoculate a flaskcontaining a suitable inoculum development medium. Thus, for example, aculture containing a Streptomyces strain, that is capable of producing aglucose isomerase enzyme preparation, is utilized to inoculate aninoculum development substrate containing an appropriate carbon source.The organism is allowed to grow in the inoculum development medium. Theincubation period may fall within a wide period of time, depending uponthe particular microorganism strain that has been selected, and, aswell, on the particular inoculum development medium that is utilized.Generally, the incubation period may last from about 4 to about 48hours. In the usual case, an aliquot, or the entire culture, is thenutilized to inoculate a still larger volume of culture medium ornutrient. This may be repeated one or more times, as necessary. Thefinal culture, which is grown in a medium specifically formulated toinduce enzyme production, is then used as the source of enzyme, with orwithout purification procedures, to effect the isomerization of glucoseto levulose.

The carbon source in the culture media may be xylose, a mixture ofxylose and starch, or xylose in combination with other carbon sourcessuch as, for example, mannitol, gluconic acid, galactose, glycerin,sorbitol, glucose, and other pure or impure sources of carbohydrates.

The xylose portion of the culture media may be xylose itself or itsnative forms that exist in cell walls of almost all plants in the formof the xylan polymer of xylose, which contains xylose as its mainconstituent. Thus straw, chaff, wood, corn cobs, wheat bran, and thelike may be employed as the source of xylan. Ordinarily, these materialsare treated with alkali to extract the xylose polymer.

A preferred source of carbon in the culture media is a mixture composedof 25 to 75 percent by weight of starch and 75 to 25 percent by weightof xylose, based on the combined weight of these two materials.Expressed in a different manner, the carbon source comprising themixture of these two materials preferably comprises from 0.2 g. to 10 g.per 100 ml. of the culture medium. More preferably, the mixture of thesetwo materials comprises from 0.5 g. to 3 g. per 100 ml. of the culturemedium.

A preferred protein source, in the culture media, is com steep liquor. Avery desirable culture medium can be made up that includes from 0.1 g.to 5 g. of corn steep liquor for every 100 ml. of the culture medium.

The culture media may also contain, in addition to the carbon source,materials such as, for example, inorganic salts such as magnesiumsulfate, potassium dihydrogen phosphate, and the like.

The addition of glycine, with or without the addition of ammoniumnitrate, is beneficial at each stage in the propagation of themicroorganism. Moreover, the addition is beneficial with all knownculture media compositions. Exemplary compositions for use inpropagating microorganisms of the Streptomyces genus, that can beimproved by the use of the present invention, would include thefollowing representative compositions:

TABLE I MEDIUM COMPOSITIONS Medium I Slant Medium for CultureMaintenance g/liter Xylose 10 Yeast Extract (Difco) 1 Beef Extract(Difco) 1 Bacto-tryptone (Difco) 2 Agar 20 CoClgGlfiO 0.24, when usedMedium ll Inoculum Development Medium g/liter Xylose 5 Corn Starch 5Corn Steep Liquor (as is) 40 MgSO 7H 0.5 CoCl 6" Q (optional) 0.24 (loM) (pl-1 adjusted to 7.1 prior to sterilization) Medium 111 FermentationMedium g/liter Xylose 10 Corn Starch l0 Corn Steep Liquor, as is 40MgSOfl up 0.5 CoCl,'6H,0(optional) 0.24 (IO- M) (pH adjusted to 7.1prior to sterilization) DETAILED DESCRIPTION OF THE INVENTION Thefollowing examples provide several demonstrations of the manner in whichthe present invention may be practiced. All references to parts and topercentages are by weight, unless expressly indicated to be otherwise,and are on an as is basis unless expressly stated to be on a dry orother basis.

EXAMPLES Production Of Glucose Isomerase Enzyme Preparation FromStreptomyces olivochromogenes ATCC 21,1 14 And The Effects Of Glycine InThe Culture Medium These examples represent practice of the presentinvention in accordance with one preferred mode thereof.

A. Inoculum Development Spores from a slant of Streptomycesolivochromogenes ATCC 21,1 14 were inoculated into several 500 ml.Erlenmeyer flasks each containing ml. quantities of a sterile mediumcomposed of the ingredients described below in Table 11.

TABLE 11 Ingredients Amounts Xylose 5.0 g. Corn Starch 5.0 g. Corn SteepLiquor (as is) 40.0 g. Magnesium Sulfate (Ms .Q4 1 29) s- CobaltChloride (c cl saw 0.24 Distilled Water 1000 ml.

The pH of the culture medium was adjusted to 7.1 with sodium hydroxideprior to sterilization. The flasks were inoculated and incubated for 60hours at a temperature in the range from about 28C. to about 30C. on areciprocating shaker.

B. Production of the Enzyme Preparation Into each of several 1,000 ml.Hinton modified Erlenmeyer flasks was placed a quantity of 200 ml. ofsterile medium of the following composition:

TABLE II Ingredients Amounm Xylose 10.0 g. Corn Starch 10.0 g. CornSteep Liquor 40.0 g. Brewers Yeast Extract 2.5 g. Maggflsliumflstgfate(M 0.5 g. Cogalt Climide C z 15th?) 0.24 s Distilled Water 1000 ml.

The pH was adjusted to 7.1 with sodium hydroxide prior to sterilization.

In preparing either of the media described above, xylan hydrolysate canbe used in place of xylose with substantially equivalent results. Thehydrolysate is an inexpensive source of xylose.

The culture media in six of the flasks (Set A in the drawings) were thentreated in the following way. To a first flask, nothing was added. Tothe other five, ammonium nitrate was added, in the following quantities,respectively: 0.1; 0.2; 0.3; 0.4, and 0.5 g., per 100 ml. of the culturemedium.

The culture media in a second set of six flasks (Set B in the drawings)were also modified, in the following way. To the first flask, nothingwas added. Amounts of glycine in the following quantities were thenadded to the remaining five flasks, respectively: 0.1; 0.2; 0.3; 0.4,and 0.5 g., per 100 ml. of the culture medium.

Set C: The procedure followed in preparing the Set B flasks was followedwith respect to the addition of glycine to the six flasks of Set C.Then, to each of these six flasks, 0.1 g. of ammonium nitrate was addedfor each 100 ml. of culture medium.

Set D: The procedure followed in preparing the Set B flasks was againfollowed. Then to each of these flasks of Set D, 0.2 g. of ammoniumnitrate was added for each 100 ml. of culture medium.

Set E: Another set of six flasks was set aside and identified as Set E.The procedure used in preparing the Set B flasks was again followed,then 0.3 g. of ammonium nitrate was added to each flask for each 100 ml.of culture medium therein, respectively.

Set F: This set of flasks was prepared in the same manner as Set E, butthe addition of ammonium nitrate to each flask in the set was 0.4 g. foreach 100 ml. of culture medium.

Set G: This set of six flasks was prepared in the same manner as the sixflasks of Set F, except that the addition of ammonium nitrate to eachflask was in the amount of 0.5 g. of ammonium nitrate for each 100 ml.of culture medium in each flask.

The contents of the several inoculum flasks were then blended togetherand mixed to insure homogeneity. Aliquot portions of ml. each were thentaken from the mixture. These aliquots were then used to inoculate eachof the flasks in each of the sets that were prepared as just described.The inoculated flasks were then incubated for 48 hours at a temperaturein the range from about 28C. to about 30C. on a rotary shaker.

C. Harvesting of the Enzyme Preparation After fermentation, an aliquotfrom each flask was centrifuged at 10,000 times gravity for minutes. Thecentrifugate was decanted off and the cell pack saved for assay. Thecell pack was either assayed immediately or, alternately, was storedfrozen until assayed.

D. Solubilization of Enzyme Immediately prior to assay, the cell packwas brought back to its original volume with 0.05 molar potassiumphosphate buffer (pH 7.5), and the cells were resuspended. Thereconstituted cell pack was then sonicated at 0-5C for 1.5 2.0 minuteswith the aid of a Branson Model W-l85D Sonifier operating at a settingof 80 watts.

The isomerase activity of the sonicated cells was then so ner:

TABLE III Component Amount 0.1 M MgSO4 7H2O 1 ml. 0,01M C0CL2 -6 H O 1ml. 1 M Phosphate Buffer, pH 7.5 0.5 ml. Anhydrous D-Glucose 1.44 g.

To make up a total Distilled Water volume of 7.5 ml.

- The solubilized enzyme preparation to be assayed was first diluted tocontain from 1 to 6 isomerase units per ml.

The enzymatic isomerization was conducted by adding 1 ml. of the enzymepreparation to 3 ml. of the stock solution, and incubating for 30minutes at 60C. At the end of the incubation period, a 1 ml. aliquot wastaken and quenched in a 9 ml. volume of 0.5 N perchloric acid. Thequenched aliquot was the diluted to a total volume of 250 ml. As acontrol, for comparative purposes, a glucose blank was also run bysubstituting 1 ml. of water for the 1 ml. of the enzyme preparation insolution form, at the beginning of the incubation period.

The ketose was then determined by a cysteinesulfuric acid method. Forthe purposes of this assay, one isomerase unit is defined as the amountof enzyme activity that is required to produce one micromole of levuloseper minute under the isomerization conditions described.

F. Tabular Presentation of the Results TABLE IV EFFECTS OF GLYCINE ANDAMMONIUM NITRATE ON THE PRODUCTION OF ISOMERASE BY Streptomycesolivochromogenes GLYCINE, grams/ ml. 0.0 0.1 0.2 0.3 0.4

Ammonium Nitrate,

gram/100 ml ISOMERASE YIELD, UNITS/ml. 0.0 3.0 4.1 4.7 4.2 7.6 8.5 0.14.0 6.0 5.8 8.8 8.9 8.2 0. 4.0 8.3 8.8 8.2 8.5 7.7 0.3 2.2 6.5 8.8 9.38.2 8.1 0.4 2.1 7.4 8.8 8.8

DRY CELL WEIGHT,

GRAMS/LITER 0.0 6.4 7.3 7 4 9.1 10.0 10.3 0.1 7.0 10.6 10.0 9.6 9.8 10.10.2 5.1 9.6 9.3 9.9 9.9 9.8 0.3 5.1 7.9 9.1 10.1 10.4 11.5 0.4 4.3 7.98.5 9.4 10.3 10.4 0.5 3.1 8.4 8.3 9.3 10.0 10.3

SPECIFIC ACTIVITY,

UNl'lS/GRAMS DRY CELL G. Graphical Representation of the Results Forbetter visualization and ease of comparison, the data of Table IV ispresented graphically in the drawmgs.

In FIG. 1., each plotted line represents a constant content level ofammonium nitrate. Each plotted point along the Set A line represents theconcentration of added ammonium nitrate. Each plotted point along eachother line represents the concentration of glycine -that has been addedto the culture medium.

Thus, for example, the plotted line for Set A represents 6 diflerentlevels of ammonium nitrate addition, beginning with zero and ending with0.5 g. per 100 ml. of culture medium, without any glycine addition atall. The plotted line for Set B records the effects of different levelsof glycine addition, in the absence of any addition of ammonium nitrate.The plotted lines for Sets C through G, inclusive, illustrate theeffects of difierent levels of ammonium nitrate addition at differentenrichment levels of glycine.

As the plotted lines in FIG. 1 demonstrate, the addition of ammoniumnitrate alone produces a slight stimulatory effect on the production ofisomerase, up to a concentration of about 0.2 g. of the ammonium nitratefor each 100 ml. of culture medium. Above this level,

the ammonium nitrate addition appears to depress the production of theenzyme.

On the other hand, the addition of glycine appears to enhance enzymeproduction when used at all the levels of addition recorded in FIG. I.

The combination of the ammonium nitrate and of the glycine exhibitsynergisn, as demonstrated in FIG. 1, in a manner that is totallyunexpected.

The economic advantage of this synergistic action is substantial, sincethe addition of ammonium nitrate reduces the amount of glycine that mustbe added to produce a given enhancement in the enzyme production. Sinceammonium nitrate is very much less expensive than glycine, there is adefinite economic incentive for use of the synergistic combinationrather than just the glycine alone.

FIG. 2 is a graphical illustration of the effect of the additions ofglycine and of ammonium nitrate on the growth of the microorganism. Itappears that the addition of the ammonium nitrate alone exhibits a toxiceffect on the growth of the microorganism, at least at concentrationsabove 0.1 g. per 100 ml. of the culture medium. As shown by the plottedline for Set B, the addition of glycine alone enhances the production ofcells.

The addition of both glycine and ammonium nitrate exhibits a synergisticeffect on the production of cells, as is shown by the plotted lines forSets C through G inclusive. This synergistic effect on cell growth ismost marked at low levels of addition of the salt and of the amino acid.The markedly enhanced growth rate also may be achieved by the additionof higher concentrations of glycine alone.

FIG. 3 is a graphical representation of the effect of the additives onthe units of the isomerase enzyme per gram of cells of themicroorganism, on a dry basis. This figure demonstrates that althoughthe cell growth is stimulated by the addition of glycine and of ammoniumnitrate, the production of the enzyme is enhanced to an even greaterdegree. This is an important and an unexpected finding, of greateconomic significance.

Comparable results are obtained when the microorganism employed isStreptomyces venezuelae ATCC 21,113.

FURTHER EXAMPLES TABLE V lngredients Amounts, g/l Xylose 20 15 DE. cornsyrup solids 1 Corn steep liquor (as is) 36 Magnesium sulfate 0 sFollowing generally the procedures already described, the data that ispresented below in Table VI was observed.

TABLE Vi Glycine added, g/ 100 Microorganism St. olivuchmmagenes ATCC 2I ,1 14

lsomerase yield, u/ml 2.2 3.0 3.1 3.6

Cell wL, g/l 6.7 6.8 7.5 7.7

Sp. act, u/g 330 440 410. i 470 St. phoechromogenes NRRL B 2119lsomerase yield, u/ml 1.3 2.4 3.0 4.1

Cell wt., gll 4.6 7.2 8.4 9.3

Sp. act., u/g 280 320 360 440 St. griseoruber lsomerase yield, u/ml 1.11.5 1.8 2.5

Cell wt., g/l 6.6 7.7 8.4 9.2

Sp. act, u/g I70 190 220 270 GENERAL The economic advantage of thepresent invention is very substantial. The cost of the culture mediumfor an industrial scale fermenter may be several thousand dollars. Thecost of adding enough glycine to have the effect of doubling the yieldof isomerase is a small fraction of the total medium cost, but candouble the enzyme yield.

We have found that when the amount of glycine that is added to theculture medium is in an amount equivalent to an addition rate of 0.1 g.to 0.25 g. for each 100 ml. of culture medium, the recovery of isomeraseenzyme is increased from 1% to 2 times. An increase in the overallamount of microbial cells produced is also observed, but the increase isnot on the same order of magnitude as the increase in enzyme production.

The amount of glycine added may be in excess of an amount equivalent to0.25 g. for each 100 ml. of culture medium. Thus, the addition of anamount equal to 0.5 g. of glycine for each 100 ml. of culture mediumwill produce more microbial cells than will a 0.25 per g. per 100 ml.addition. However, the increased amount of enzyme produced will be lessthan that produced in the culture medium when a smaller amount ofglycine is added. Moreover, it has been found that when the rate ofglycine addition is equivalent to 1.0 g. per 100 m1. of culture medium,growth inhibition occurs and the recovery of enzyme is less than that ata rate of glycine addition equivalent to 0.5 g. per 100 ml. of culturemedium.

Although certain other amino acids do have some stimulating effect onproduction of enzyme, the effect is generally subtantially less thanthat obtained with glycine, or requires so much of the amino acid as tobe impractical.

The addition of glycine to the culture medium, either alone or incombination with ammonium nitrate, appears to be generally useful forenhancing isomerase production with all known microorganisms thatproduce the isomerase. Thus, the demonstrations of the invention thathave been described can be substantially duplicated with other strainsof Streptomyces, such as, for example, S. venezuelae ATCC 21,113.Enhanced enzyme production can also be obtained through the use ofglycine in the culture medium, with or without the addition of ammoniumnitrate, in the production of many other Streptomyces species. Forexample, the following are representative:

S. achromogenus S. griseolus S. albus S. gn'seoruber S. antibioticu:ATCC 10,332 S. griseus S. aureus S. herbarioolor S. bobiliae S.hydroscapicu S. califomicus S. Iipmam' S. caelicolor S. m'veoruber S.diaslaticur S. alivaceu: S. flavoviren: S. phaeochromogenes' S. fmdiaeS. pur ofusclu S. gaelicolor S. me i S. galbus S. roseachromogener S.galilaeus S. rulgersensis' S. gedanensis In addition, many differentsoil isolates produce the desired enzyme. The present invention isefficacious for use with such microorganisms, and, as well, with suchother diverse enzyme-producing producing organ isms as: Pasteurellapestis; Lactobacillus pentosus; L. brevis; L. gayonii; L. fermenti; L.pentoaceticus; L. fermentum; L. lycopersici; L. buchneri; and L.xylosus; strains of Bacillus isolated from laboratory air; B.megaterium; Aerobacter cloacae; A. aerogenes; Candida utilis;Escherichia intermedia; Micromonospora; Mycobacterium phylei; M.Smegmatis; Actinomyces fluorescens; Brevibacterium PentosoAminoacidicum; and Leuconostic mesemeroides.

In addition, enhanced enzyme production is also observed by using thetechniques of the present invention in the culture of thermophilicBacilli, particularly Bacillus stearothermaphilus.

Enzymes produced in accordance with the present invention are useful, ofcourse, in the production of levulose-bearing products from starchhydrolyzates and dextrose solutions.

CONCLUSION While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications, and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice in the artto which the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth, and as fall within the scope of theinvention.

We claim:

1. A method for the culture of microorganisms which produce glucoseisomerase comprising culturing said microorganisms in a mediumcomprising from 0.1 g. up to 0.5 g. of glycine/ 100 ml. of culturemedium, from 0.1 g. up to 0.5 g. of ammonium nitrate/100 ml. of culturemedium and xylose as a carbon source.

2. The method of claim 1 wherein the microorganism is a Streptomycesmicroorganism.

3. The method of claim 1 wherein the microorganism is Streptomycesolivochromogenes ATCC 21,1 14.

4. The method of claim 1 wherein the microorganism is Streptomycesvenezuelae ATCC 21,113.

5. The method of claim 1 wherein the culture medium contains from 0.2 g.to 10 g./ 100 ml. of culture medium of a mixture composed of 25 topercent by weight of starch and 75 to 25 percent by weight of xylose.

2. The method of claim 1 wherein the microorganism is a Streptomycesmicroorganism.
 3. The method of claim 1 wherein the microorganism isStreptomyces olivochromogenes ATCC 21,114.
 4. The method of claim 1wherein the microorganism is Streptomyces venezuelae ATCC 21,113.
 5. Themethod of claim 1 wherein the culture medium contains from 0.2 g. to 10g./100 ml. of culture medium of a mixture composed of 25 to 75 percentby weight of starch and 75 to 25 percent by weight of xylose.